Antibiogram and virulence profiling reveals multidrug resistant Staphylococcus aureus as the predominant aetiology of subclinical mastitis in riverine buffaloes

Abstract Background Staphylococcus spp. are the major causal agents of mastitis in dairy animals worldwide leading to profound economic losses and public health threats. Recently, Staphylococcus aureus has emerged as a multidrug resistant and zoonotic pathogen. This study aimed to characterize S. aureus in subclinical mastitis (SCM) milk samples of riverine buffaloes in Bangladesh through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing. Method We characterized S. aureus in SCM milk samples (N = 500) of riverine buffaloes through antibiogram and virulence gene(s) profiling, and 16S rRNA gene sequencing. Results Out of 500 milk samples tested, 188 (37.6%) were found positive for SCM. From 188 SCM samples, 291 isolates were obtained with a prevalence of S. aureus in 37.4% (109/291) isolates. Phylogenetic analysis revealed the evolutionary divergence of S. aureus isolates in bubaline SCM milk samples. The antibiogram profiling showed that about 96.0% S. aureus isolates were multidrug resistant (MDR). Notably, 29 and 16 isolates harboured methicillin‐resistant (mecA) and panton‐valentine leucocidin (pvl) genes, respectively, and 46 plasmid‐bearing isolates were MDR. Nine Staphylococcal enterotoxins (SEs/SEls) including sea (11.9%), sec (7.4%), sed (4.6%), seg (3.7%), and seh (3.7%) were detected with 72.48% toxinotypes comprising a single gene. Conclusion This study therefore suggests S. aureus as the single‐most aetiology (∼37.0%) of SCM in riverine buffaloes, and emergence of MDR, enterotoxin producing, and virulent S. aureus strains could impose potential threats to animal welfare and public health.


INTRODUCTION
staph" or "superbug" are a major cause of nosocomial infections that lead to increased mortality rates and a substantial increase in human health care costs (Shrestha et al., 2021). Staphylococcus aureus is an increasingly recognized etiologic agent of bovine (Hoque et al., 2018;Rossi et al., 2019) and bubaline (de Medeiros et al., 2011;Guccione et al., 2020) mastitis and has been remaining as the most frequently isolated bacterium from udder infections especially in herds with high prevalence of SCM. Furthermore, S. aureus is categorized as one of the major pathogens causing mastitis in dairy animals (El-Ashker et al., 2015) and responsible for about one-third of cases of the SCM (Hoque et al., 2018;Haran et al., 2012;Shome et al., 2011). The ability of S.
aureus to cause mastitis is probably due to the expression of various toxins, virulence factors, and cell wall adhesion proteins (El-Ashker et al., 2015;Haran et al., 2012). The bacterium has the ability to survive phagocytosis in the udder and often causes chronic inflammation (El-Ashker et al., 2015;Hoque et al., 2018). Therefore, early diagnosis of SCM is essential because changes in udder tissue take place much earlier than they become apparent.
The resistance of S. aureus to commonly used antibacterial agents presents an increasing challenge and can complicate the treatment of mastitis. High prevalence of antimicrobial resistance (AMR) in both animal and human S. aureus isolates could be due to widespread, uncontrolled, and indiscriminate use of antibiotics (Al Amin et al., 2020Amin et al., , 2022. The use of broad-spectrum antibiotics creates a selective pressure on the bacterial flora, thus increasing the emergence of new antibiotic-resistant bacteria (Bantawa et al., 2019;Hoque, Istiaq, Clement, et al., 2020). Although the prevalence of MRSA strains in mastitis seems to be generally low in buffalo farms (El-Ashker et al., 2015), the emergence of antibiotic-resistant microorganisms poses a potential public health hazard. More recently, multidrug resistance (MDR) was detected in both coagulase-positive and coagulasenegative Staphylococci isolated from buffaloes and cows mastitis (Aires-de-Sousa et al., 2007;Dorgham et al., 2013). Furthermore, the treatment efficacy of S. aureus mastitis is usually disappointing because of excessive damages in the mammary tissues and drugs are not able to penetrate to all infected sites (Bantawa et al., 2019;Hoque, Istiaq, Clement, et al., 2020). This bacterium also suppresses phagocytosis and cell-mediated immunity and produces an enzyme that inactivates most of the antimicrobial agents (De Oliveira et al., 2000;Hoekstra et al., 2020).
The need for reliable and rapid methods for identification of S. aureus is crucial for the control of the disease and for economically sound udder health management. The identification and characterization of Staphylococci can be performed using various phenotyping and genotyping techniques such as biochemical investigations, molecular assays including polymerase chain reaction (PCR), DNA sequencing (ribosomal gene and complete genome sequencing), and physical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Bianchi et al., 2014;Barreiro et al., 2017;Phuektes et al., 2003). Molecular methods provide accurate confirmation of the identity of microorganism isolated from mastitis (SCM or CM) milk samples (Annamanedi et al., 2021;Hoque et al., 2018). In most laboratories of Bangladesh, characterization of Staphylococci is done by the identification of phenotypic traits of cultured bacteria, which sometimes provide confusing results. Again, it needs sufficient time for bacterial growth in culture medium. Conversely, PCR-based approach is a rapid, efficient and cost-effective tool for accurate characterization of the causal agents (Bianchi et al., 2014). A series of reports are available describing the use of PCR and ribosomal (16S rRNA) gene sequencing to identify and characterize Staphylococcal isolates (Bianchi et al., 2014;Phuektes et al., 2001;Shome et al., 2011). Moreover, multiplex PCR (mPCR) assay for simultaneous detection of SCM causing pathogens could be a promising tool for monitoring mastitis at the herd level (Ashraf et al., 2017;Charaya et al., 2015). For rapid and reliable detection of S. aureus and its enterotoxins in milk, a number of mPCR assays have been established (Ashraf et al., 2017;Rahman et al., 2018;Charaya et al., 2015).
Buffalo in Bangladesh is mainly indigenous in origin and most of them are riverine type with exception of some swamp type in eastern part of the country (Hoque et al., 2011 Figure S1). The buffaloes were of 4 to 9 years of age, 2.5-3.5 body condition score (BCS), and 2-4 parity. The aver-age milk production was 7.5 L per day. Among these, 75 buffalo cows belonged to smallholder farms, whereas 50 cows belonged to two commercial farms. Due to the absence of observable clinical signs, presumptive diagnosis of SCM was made by California Mastitis Test (CMT) following our previously published protocol (Hoque et al., 2015) and manufacturer's instruction (CMT ® , Original Schalm reagent, Thech-niVet, USA) ( Figure S2). Buffalo cows showing clinical signs of mastitis were excluded from the study.

Collection of milk samples and bacteriological examination
Five hundred quarter milk samples were collected from 125 riverine buffalo cows. Before collection, quarters were soaked with 70.0% ethanol and dried off using tissue paper. Initially, one to two drops of milk were discarded and then 10-15 ml of milk from each of the four quarters (left front; LF, left hind; LH, right front; RF, and right hind; RH) was collected aseptically in sterile plastic tubes (15 ml Falcon Tube ® ).
Immediately after collection, milk samples were subjected to CMT to diagnose quarters with SCM ( Figure S2). After on farm screening, the SCM-positive samples (n = 188) were kept in icebox (4-8 • C) and transported to the laboratory for further bacteriological examination and molecular analysis.

DNA extraction from S. aureus isolates
Genomic DNA was extracted from colonies of overnight cultures with typical growth of Staphylococci on Mannitol salt agar (MSA, Oxoid, Hampshire, UK) using the boiled method (Hoque, Istiaq, Clement, et al., 2020). In brief, pure bacterial culture from nutrient agar was subcultured in nutrient broth. Each millilitre broth culture was taken in separate Eppendorf tube and centrifuged at 10,000 rpm for 5 min. The supernatant was discarded and any remaining liquid was removed by soaking (with wipes). The pellet was collected and replenished with 200 µl autoclaved deionized water followed by finger shaking to dissolve the pellet. The Eppendorf tubes were then placed in a water bath at 100 • C for 10 min. Immediately after boiling, the Eppendorf tube was kept in ice for 10 min followed by centrifugation at 10,000 rpm for 10 min. Finally, about 100-150 µl supernatant containing bacterial chromosomal DNA was collected. DNA quantity and purity were determined with NanoDrop 2000 (ThermoFisher, USA) by measuring 260/280 absorbance ratios, and stored at −20 • C.

PCR amplification, 16S rRNA gene sequencing, and data analysis
Following DNA extraction, PCR amplification was carried out targeting the ribosomal (16S rRNA) gene fragments. The 16S rRNA gene was amplified using universal primers 27F (5t'-AGAG TTTGATCCTGGCTCAG-3t') and U1492R (5t'-CTACGGCTACC TTGTTACGA-3t') (Masomian et al., 2016). Agarose gel electrophoresis (1.2% wt/vol) was used to verify the presence of PCR products. DNA purification and standard Sanger sequencing were conducted for five isolates following the instructions of the sequencing company (Macrogen Inc. Seoul, Korea). Using Molecular Evolutionary Genetics Analysis (MEGA) version 7.0 for the larger datasets (Kumar et al., 2016), the nucleotide sequence of the corresponding isolates was visualized. In order to search for nucleotide sequences similarity, Genbank databases were used by online program nucleotide BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) (Donkor et al., 2014).
Closely related sequences were retrieved from NCBI and subjected to multiple sequence alignment by ClustalW program (Thompson et al., 2003). Trimmomatic program (version 0.39) was used to estimate the quality of each sequence, edit and trim poor-quality sequences (Rahman et al., 2021). A maximum-likelihood tree was generated by MEGA 7.0 software using default parameters, and visualized by iTOL v5.6.1 (Letunic & Bork, 2019). Nodal confidence in the resulting phylogenetic relationships was assessed using the bootstrap test (1000 replicates). The sequences were submitted in NCBI (https://www.ncbi.nlm.nih.gov), with the accession numbers from ON386175 to ON386179.

Antibiogram profiling
The in vitro antibiogram profile of 109 S. aureus isolates was determined using the disk diffusion method following the Clinical Laboratory Standards Institute (CLSI) 2017 guidelines (Arendrup et al., 2017).

PCR amplification of tsst-1 and pvl toxin encoding genes in S. aureus isolates
The genomic DNA extracted from the S. aureus isolates (as mentioned above) was used to screen for the toxic shock syndrome toxin-1 (tsst-1) gene and the Panton-Valentine leukocidin (pvl) toxin gene following previously described protocols (Haran et al., 2012).
PCR products were electrophoresed in a 1.5% agarose gel containing 500 µg ml -1 of ethidium bromide, and the gel was visualized by UV transilluminator.

Multiplex PCR for detection of enterotoxin (SEs/SEls) genes in S. aureus isolates
A modified mPCR was used for the detection of enterotoxin genes from the S. aureus isolates (Bianchi et al., 2014;Tarekgne et al., 2016). In brief, each mPCR reaction was performed with a final reaction volume of Imaging System (Bio-Rad, USA). The S. aureus isolates were investigated for the presence of genes coding for nine selected SEs (sea, seb, sec, sed, see, seg, seh, sei, ser) and two SEls (selj, selp) according to the recommended guidelines of the European Union Reference Laboratory for coagulase-positive Staphylococci by using two independent mPCR reaction protocols (Bianchi et al., 2014). Primers for sed, see, seg, sei, and tsst-1 were combined in reaction mixture 1 and primers for sea, seb, sec, seh, sej, and 16S rRNA were combined in reaction mixture 2 (Carfora et al., 2015). The primers used were supplied by Invitrogen Life Technologies (Carlsbad, CA, USA) and listed in Table   S1. Each S. aureus isolate positive for at least one of the "classical SEs" coding gene by mPCR was tested for Staphylococcal enterotoxins production (SEA-SED), performed by the RPLA method (Carfora et al., 2015), using the kit SET-RPLA (TD 9000; Oxoid, Hampshire, UK) according to the manufacturer's instructions.

Plasmid extraction
All of the S. aureus isolates were further tested for the presence of plasmid. Plasmid DNA from S. aureus isolate was extracted according to previously published methods (Hoque et al., 2018;Tumlam et al., 2013 For the test, p < 0.05 was considered statistically significant.

Prevalence and aetiology of subclinical mastitis
A total of 500 quarter milk samples from 125 riverine buffalo cows were screened through CMT to diagnose buffalo cows with SCM. The  (Table 1). In our present study, there were marked differences in the prevalence of S. aureus in the SCM milk across the study TA B L E 1 The relative distribution of bacterial aetiologies identified from quarter milk samples of buffalo cows infected with subclinical mastitis  (15), Staphylococci + E. coli (11), Staphylococci + Bacilli (10), Streptococci + E. coli (9), Streptococci + CNS (7).

Subclinical mastitis associated S. aureus showed multidrug resistance properties
Staphylococcus aureus revealed varying susceptibility to 15 antibiotics belonging to nine different groups ( Table 2). Out of 109 S. aureus isolates tested, 105 (96.33%) isolates were resistant to antibiotics belonging to more than one group of antibiotics tested and were classified as MDR isolates (

Prevalence of mecA, pvl, and enterotoxin (SE/SEl) genes
The toxin gene profile of S. aureus isolates recovered from SCM milk samples of buffalo cows is listed in

Plasmid bearing S. aureus isolates showed multidrug resistance
Plasmid extraction from S. aureus isolates revealed that 70.6% (77 out of 109) isolates were harbouring one or more plasmids. From the result of antimicrobial susceptibility test, it has been found that 42.2% (46/109) of these plasmid bearing isolates were MDR isolates. The remaining 31 plasmids were susceptible to all the tested antibiotics.
The size of the plasmids was found to be >10 kb. No plasmid DNA was found in 29.4% (32/109) isolates, and among these isolates, 19 (59.4%) were resistant to at least three or more antimicrobials.

DISCUSSION
Subclinical mastitis is considered one of the most infectious diseases that affect dairy animals with an annual prevalence of 37% (El-Ashker et al., 2015). Although epidemiological prevalence of S. aureus and its antimicrobial resistance pattern have been extensively studied in livestock in other countries (Cheng et al., 2019;El-Ashker et al., 2015;Zayda et al., 2020), limited studies with particular emphasis to SCM in lactating dairy cows and buffaloes have been carried out in Bangladesh. Therefore, this study aimed to characterize S. aureus isolates in milk obtained from riverine buffalo cows affected with SCM to their antimicrobial resistance and virulence gene(s) under the current farming situation of Bangladesh. Moreover, the evolutionary relationship between the strains isolated from bubaline SCM milk and those retrieved from the global database (NCBI) belonging to cow and buffalo milk was also investigated (Figure 1).
The prevalence of SCM in riverine buffaloes was 37.6% which is consistent with many of the studies from our neighbouring countries India (25.0%-35.0%) (Krishnamoorthy, Goudar, et al., 2021;Srinivasan et al., 2013) and Pakistan (35.0%-45.0%) (Hussain et al., 2018;Krishnamoorthy, Goudar, et al., 2021). with the other studies conducted in various neighbouring countries (Ali et al., 2021;Pankaj et al., 2013), and elsewhere in the world Osman et al., 2014). However, mixed infections were found in 29.5% culture-positive milk samples and of them, Staphylococci and Streptococci were the most common mixed causes of bubaline SCM (∼29.0%). Similar to our findings, other researchers have also reported S. aureus as the main etiological agents of mastitis in different parts of India (Neelesh et al., 2010;Pankaj et al., 2013). Contrary to our findings, several earlier studies (Pizauro et al., 2019;Sampimon et al., 2009) reported a lower prevalence of S. aureus and a relatively higher prevalence of the environmental pathogens, such as coagulase-negative Staphylococci (Zayda et al., 2020), which are formally described as a minor pathogen of mastitis .
In our present study, there were marked differences in the prevalence of S. aureus in SCM milk samples collected from different study areas, seasons, and quarters of the buffalo cows. The highest prevalence of S. aureus in SCM milk was found in Bhola (55.6%) and the lowest in Mymensingh (33.1%). Consistent with our findings, researchers from other countries have also reported that the prevalence of S. aureus in mastitis milk could vary in different study locations (Cheng et al., 2019;Neelesh et al., 2010;Pankaj et al., 2013;Pizauro et al., 2019).
The quarter-wise prevalence of S. aureus in SCM milk remained highest in right rear (44.5%) quarters and lowest in right front (25.4%) quarters. Similar prevalence of SCM in buffalo cows has been reported in India (Pankaj et al., 2013;Sharma & Maiti, 2010). The difference in prevalence of SCM and its associated pathogens found in the present and the previous studies might be because the bacteria causing mastitis are changing with topographical and management conditions. The phylogenetic tree also exhibited different lineages of aureus strains of different countries of the world (Figure 4). This result corroborates with our previous study in Bangladesh (Hoque, Istiaq, Clement, et al., 2020) where we reported a genetic similarity between Bangladeshi S. aureus isolates and S. aureus strains of the other countries of the world. Furthermore, several studies indicated that genetic dimension among the isolates of the world is extremely relative, and 16S rRNA analysis is considered a good discrimination approach for distinguishing unrelated isolates (Gumaa et al., 2021). This was in partial agreement with the study of Ali et al. (2021). They also reported that oxytetracycline, ampicillin, and doxycycline were the most resistant antimicrobials against major mastitis pathogens like S. aureus (Ali et al., 2021 (Bantawa et al., 2019;Hoque, Istiaq, Clement, et al., 2020;Hoque et al., 2018;Shrestha et al., 2021 (Algammal et al., 2020;Hoque, Istiaq, Clement, et al., 2020;Shrestha et al., 2021 (Elsayed et al., 2015;Carfora et al., 2015;Hoque et al., 2018). Moreover, sea and seg (Carfora et al., 2015;Martins et al., 2015;Wang et al., 2009), and sec, sed, seg, and sei (Hoque et al., 2018;Martins et al., 2015;Zecconi et al., 2006) are the genes most frequently detected in S. aureus isolated from mastitis suffering dairy animals.
Plasmids are key reservoirs for genetic content in Staphylococci and allow the rapid propagation of antibiotic resistance (Mores et al., 2021). Because of the limitations we faced with fewer samples, it would be interesting to conduct similar trials using a larger sample size with a different animal population (breed, parity, body condition, lactation) and matrices prior to undertaking a further genomic diagnostic venture to elucidate the molecular mechanisms and mutational spectra in the genome or pangenomes of S. aureus to better understand the pathophysiology mastitis. Another important limitation we faced in the present study was the farmers unawareness regarding the prevalence of SCM in their buffalo cows (since there were no presenting sign/clinical symptoms), and in most of the cases, SCM in buffalo cows remained undiagnosed or unattended. These limitations on mastitis in riverine buffaloes in Bangladesh are a serious problem because the inability to correctly and accurately identify the pathogen leads to difficulty in selecting the appropriate pathogen-specific treatment or control measure to apply, consequently increasing development, and the rapid emergence of multidrug resistant S. aureus strains.

CONCLUSIONS
Staphylococcus aureus remains as the primary pathogen of concern in cases of SCM in riverine buffaloes in Bangladesh. The higher prevalence of S. aureus (>37.0%) in relation to other microbes (both major and minor pathogens) causing SCM in buffaloes in Bangladesh indicates a serious public health problem. Phylogenetic analysis reveals evolutionarily diverse origin of S. aureus isolated from SCM milk of buffaloes, and shows close relationship with many pathogenic strains of S. aureus of both animals and humans reported from other countries of the world. The antibiotic sensitivity tests revealed S. aureus as an MDR pathogen carrying both mecA and pvl genes along with nine different Staphylococcal enterotoxins (SEs/SEls). With limited molecular data currently available, our study provides crucial background information for future researchers and could help elucidate the epidemiological spread of S. aureus, the underlying molecular mechanisms, and mutational spectra in the genome or pangenomes of S. aureus to better understand the pathophysiology mastitis and devise optimal therapeutic strategies to curb the spread of AMR and virulence gene(s). like to thank Wali Ahad Setu and Md. Sharif Ahmed former MS students, GOR, BSMRAU for their support in the experiment. Moreover, the support and cooperation from the local Veterinarians, technicians in selection of the study farms and buffalo farmers for subjecting their cows as study materials are also acknowledged.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.942

ETHICS STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. All efforts were made considering the welfare of the experimental animals.